Human Growth and Differentiation Factor GDF-5

ABSTRACT

This invention relates to the production and use of pharmaceutical growth factor compositions with novel characteristics, e.g. improved solubility and controlled release characteristics under physiological conditions. Said compositions of one or more precursor proteins of growth factors of the GDF family provoke morphogenic effects such as for example growth, differentiation, protection and regeneration of a variety of tissues and organs, e.g. bone, cartilage, tendons, ligaments, nerves and skin. The invention can be advantageously used for the healing of tissue-destructive injuries and for the prevention or therapy of degenerative disorders.

This invention relates to the production and use of pharmaceuticalgrowth factor compositions with novel characteristics, e.g. improvedsolubility and controlled release characteristics under physiologicalconditions. Said compositions of one or more precursor proteins ofgrowth factors of the GDF family provoke morphogenic effects such as forexample growth, differentiation, protection and regeneration of avariety of tissues and organs, e.g. bone, cartilage, tendons, ligaments,nerves and skin. The invention can be advantageously used for thehealing of tissue-destructive injuries and for the prevention or therapyof degenerative disorders.

A growth factor with a variety of biological attributes isgrowth/differentiation factor 5 (GDF-5). The protein is also known asMP52, very close relatives of GDF-5 with overlapping biologicalfunctions and extremely high amino acid homologies are GDF-6 and GDF-7.The GDF-5/-6/-7 group is conserved among vertebrate/mammalian speciesbut does not have known orthologues in invertebrates (Ducy and Karsenty2000, Kidney Int. 57, 2207-2214). In general, GDF proteins promote cellproliferation and differentiation as well as tissueformation/regeneration and are relevant for a wide range of medicaltreatment methods and applications. These dimeric molecules act throughspecific receptor complexes that are composed of type I and type IIserine/threonine receptor kinases. The receptor kinases subsequentlyactivate smad proteins, which then propagate the signals into thenucleus to regulate target gene expression. Smad independent signallingpathways are also initiated by these receptors and result in inductionof the MAP Kinase pathway. Smads are a unique family of signaltransduction molecules that can transmit signals directly from the cellsurface receptors to the nucleus, where they regulate transcription byinteracting with DNA binding partners as well as transcriptionalcoactivators and corepressors.

The members of this protein family are initially synthesized as largeprecursor proteins which subsequently undergo proteolytic cleavage at acluster of basic residues approximately 110-140 amino acids from theC-terminus, thus releasing the C-terminal mature protein parts from theN-terminal prodomain. All mature polypeptides are structurally relatedand contain a conserved bioactive domain comprising six or sevencanonical cysteine residues which are responsible for thethree-dimensional “cystine-knot” motif of these proteins.

Within the mammalian body, endoproteolytic cleavage takes primarilyplace in the trans-Golgi network. The process finally leads to thesecretion of active mature protein parts, whereas the source material aswell as the prodomain portion of the cleaved precursor protein isbelieved to remain in the Golgi compartment.

Pharmaceutical compositions comprising biologically active mature GDF-5related proteins have already been developed (see e.g. WO96/33215).Mature GDF-5 has been identified to be a very effective promoter ofbone, cartilage and connective tissue formation (see for example WO95/04819, Hötten et al. 1996, Growth Factors 13, 65-74; Storm et al.1994, Nature 368, 639-643; Chang et al. 1994, J. Biol. Chem. 269,28227-28234). It is also beneficial for promoting regeneration ofvarious tissues and organs. GDF-5 is a growth factor in the nervoussystem and supports the survival of e.g. dopaminergic neurons (see forexample WO 97/03188; Krieglstein et al., (1995) J. Neurosci Res. 42,724-732; Sullivan et al., (1997) Neurosci Lett 233, 73-76; Sullivan etal. (1998), Eur. J. Neurosci 10, 3681-3688). The protein allows tomaintain nervous function or to retain nervous function in alreadydamaged tissues. GDF-5 is therefore considered to be a generallyapplicable neurotrophic factor. It is also useful for the treatment anddiagnosis of skin related disorders (WO 02/076494; Battaglia et al.2002, Trans. Orthop. Res. Soc. 27, 584), and for induction ofangiogenesis (Yamashita et al. 1997, Exp. Cell Res. 235, 218-26).

Especially the osteogenic properties of GDF-5 have been successfullyused in the past, i.e. to aid the healing of local bone fractures. Forsuch purposes, combined osteoinductive materials consisting of matureGDF-5 and solid carrier matrices have been developed (see for exampleWO98/21972). However, solid materials are inappropriate for indicationssuch as i.e. osteoporosis which require a systemic application in orderto guarantee a homogeneous distribution of the protein within the body.Likewise problematic is a drug delivery to places which are badlyaccessible such as the brain or the spinal cord.

In these cases, administration of GDF-5 in soluble form is generallypreferred. However, the mature protein shows exceptional poor solubilityunder physiological conditions. According to this fact, previousattempts to formulate stable liquid or gel-like GDF-5 compositions havefaced serious problems. A pH-dependent solubility profile of matureGDF-5/MP52 (shown i.e. in EP 1 462 126) reveals that the protein startsprecipitating in aqueous solutions with a pH above 4.25 and becomesalmost insoluble between pH 5 and pH 9. Although EP 1 462 126 succeededin improving the protein solubility profile slightly by using solventswith low ionic strength, high solubility at nearly neutral pH has neverbeen achieved but is very desirable for parenteral and otherformulations.

After discovery of their unique tissue inductive activities, growthfactor proteins such as GDF-5 have been successfully applied intherapeutic research and regenerative surgery, in which they promote andassist the natural healing process of damaged tissues, either alone orin combination with specific matrix materials. Nevertheless there isstill a great need to develop novel methods and pharmaceuticalcompositions for the efficient administration of such proteins underphysiological conditions, e.g. in cases which do not allow thecombination of the protein with a voluminous solid carrier material.Especially desirable are formulations from which the active protein isreleased in a controlled manner which exactly satisfies the demand ofthe body.

It is therefore an object of the invention to improve and facilitate themedical use of GDF-5 and related proteins by providing liquid growthfactor compositions which are stable, non-toxic and applicable atphysiological pH values. This object comprises the development ofinjectable and/or parenteral formulations, controlled releasecompositions, and formulations which can be transported across theblood-brain barrier. A second object of the invention is a method forthe preparation of said formulations and compositions. A third object ofthe invention is to provide suitable methods for the local or systemicadministration of said growth factor compositions.

These objects are solved according to the invention by providingpharmaceutical compositions containing biologically inactive precursorproteins related to human Growth/Differentiation Factor 5 (hGDF-5).These pharmaceutical compositions are intended to serve as controlledrelease formulations which are delayed activated inside the mammalianbody, either by endogenous proteases at the target site or byco-administered proteolytic enzymes.

Some frequently used terms herein are defined and exemplified asfollows:

The term “cystine-knot-domain” as used herein means the well known andconserved cysteine-rich amino acid region which is present ingrowth/differentiation factors (GDFs) and which forms athree-dimensional protein structure known as cystine-knot. In thisdomain, the respective location of the cysteine residues to each otheris important and is only allowed to vary slightly in order not to losethe biological activity. Consensus sequences for cystine-knot domainsare known in the state of the art. According to the definition definedherein the cystine-knot-domain of a protein starts with the firstcysteine residue participating in the cystine-knot of the respectiveprotein and ends with the residue which follows the last cysteineparticipating in the cystine-knot of the respective protein. Forexample, the cystine-knot domain of the human GDF-5 full length(precursor) protein (SEQ ID NO 1) comprises the amino acids 400-501 (seealso FIG. 1).

The term “precursor protein” as used herein means a biologicallyinactive protein comprising a protease site, said site being necessaryfor proteolytic cleavage of said precursor protein and subsequentlyleading to the release of a biologically active mature protein.

The term “GDF-5 related precursor protein(s)” as used herein means anynaturally occurring mammalian or artificially created, biologicallyinactive precursor protein which comprises a) a protease site which isnecessary for proteolytic cleavage of said precursor protein,subsequently leading to the release of a biologically active matureprotein, and b) a cystine-knot-domain with an amino acid identity of atleast 70% to the 102 aa cystine-knot domain of human GDF-5 (amino acids400-501 of FIG. 1/SEQ ID NO 1).

The percentage of identical residues can be easily determined byalignment of two sequences as described hereinafter, followed bycounting of the identical residues. This term includes proteinsbelonging to the group of GDF-5, GDF-6 and GDF-7 precursor proteins fromeach mammalian species as well as recombinant variants thereof as longas these proteins fulfil the above mentioned requirements. Non-limitingexamples of GDF-5 related precursor protein are precursor proteins ofhuman GDF-5 (disclosed as MP52 in WO95/04819 and in Hötten et al. 1994,Biochem. Biophys Res. Commun. 204, 646-652), recombinant humanGDF-5/MP52 (WO96/33215), mouse GDF-5 (U.S. Pat. No. 5,801,014), CDMP-1(WO96/14335), HMW human MP52s (WO97/04095), human GDF-6 (U.S. Pat. No.5,658,882), mouse GDF-6 (NCBI accession no NP_(—)038554), GDF-6/CDMP-2(WO96/14335), human GDF-7 (U.S. Pat. No. 5,658,882), mouse GDF-7 (NCBIaccession no AAP97721), GDF-7/CDMP-3 (WO96/143335), monomeric GDF-5, -6and -7 (WO 01/11041 and WO99/61611).

The term “variant(s)” as used herein means any of the followingpolypeptides:

a) fragments of said protein comprising at least the cystine-knot domainand the protease site necessary for proteolytic activation.

b) protein constructs which contain additional sequences in excess tothe original sequence of said protein

c) any combination of a) and b)

The term “biological activity” denotes the biological activities of aGDF-5 related protein or GDF-5 related precursor protein. For example,this activity can be measured by one or more of the following assays:

a) Osteogenic and chondrogenic activity can be measured by an in vitroalkaline phosphotase assay (ALP), e.g. as described in Takuwa et al.(1989), Am. J. Physiol. 257, E797-E803). This is the most useful andpreferred in vitro test, which is demonstrated hereinafter in example 4.Mature growth factors have been shown to increase alkaline phosphataseactivity i.e. in ROB-C26 osteoprogenitor cells (Yamaguchi et al. 1991,Calcif. Tissue Int. 49, 221-225) as described in WO95/04819, inembryonic ATDC5 cells (Riken Gene Bank, ROB 0565), in mouse stromalMCHT-1/26 cells, and in periodontal ligament (HPDL) cells as shown inNakamura et al. 2003, J. Periodontal Res. 38,597-605.

b) Neurotrophic activity can be determined by increased survival ofdopaminergic neurons as described for example by Krieglstein et al. 1995(J. Neuroscience Res. 42, 724-732) or Sullivan et al. 1997 (NeuroscienceLetters 233, 73-76);

c) the outgrowth of nerve fibers can be measured from embryonic retinaas described i.e. in WO97/03188;

d) the angiogenic potential of these proteins can be determined forexample in an in vivo corneal micropocket model as described inYamashita et al. 1997 (Exp. Cell Research 235, 218-226);

e) effects of GDF-5-related proteins on the terminal differentiation ofmyoblasts is described e.g. by Inada et al 1996 (Biochem Biophys ResCommun. 222, 317-322);

f) in vivo tests measuring the inductive potential of such proteinsconcerning tendon and ligament e.g. are disclosed in Wolfman et al.1997, J. Clin. Invest. 100, 321-330;

g) measurement of the signal transduction cascade through the activationof Smads using a reportergene assay based on the Smad-binding-elementspreceding the firefly luciferase gene e.g. are previously described inNohe et al., 2002. J Biol Chem. 277, 5330-5338.

Unlike their mature counterparts, precursor forms of GDF-5 relatedproteins are biologically inactive regarding their growth anddifferentiation capabilities. In addition, efficient production of theseprotein precursors in prokaryotic hosts failed seriously in the past dueto unknown reasons, although in contrast production of the significantlyshorter mature proteins is possible and has been achieved previously(see e.g. Hötten et al., Biochem Biophys Res Comm 204, 646-652 (1994).Because of these two facts, addition of GDF-5 related precursors topharmaceutical compositions instead of mature proteins never seemed tobe a reasonable option in the past.

According to the present invention, it has now surprisingly been found(and it is subsequently demonstrated hereinafter) that specific sequencemodifications in fact allow for the recombinant expression of GDF-5related precursor proteins in prokaryotic hosts, a process which iseconomically desirable.

It is furthermore shown that these recombinant proteins, althoughexpressed in bacteria and therefore lacking essential eukaryoticfeatures such as e.g. glycosylation, can be proteolytically cleaved andactivated by selected proteases in a manner comparable to glycosylatedeukaryotic precursor proteins. It is also demonstrated that theserecombinant precursor forms, unlike their mature counterparts, aresoluble at physiological pH values and can be used to formulatepharmaceutical compositions for the therapy of tissue destructivedisorders. It is finally substantiated that the disclosed pharmaceuticalcompositions comprising said precursor molecules might be beneficiallyutilized as initially inactive controlled release formulations. Theseformulations can be parenterally administered and are subsequentlyactivated in situ.

It is therefore a first object of the present invention to providesuitable methods for the heterologous recombinant expression of GDF-5related precursor proteins in prokaryotic host cells such as e.g. E.coli. Such prokaryotic production is cost effective, efficient and ofconsiderable commercial interest. Although it is known that mature GDF-5related proteins are recombinantly producible in bacteria without majorproblems, similar attempts to manufacture precursor molecules of theseproteins in sufficient quantities in E. coli failed in the past. It hasbeen postulated previously that the prodomain of the precursor proteinmay comprise partial sequences which are deleterious or even toxic forbacteria. However, according to the present invention, this explanationis no longer acceptable because it is shown hereinafter that prokaryoticexpression of the complete precursor sequence is achievable if certainamino acids are added to the original amino acid sequence. Moreprecisely, an amino-terminal sequence extension of at least five,preferably six or seven amino acids, is sufficient to allow bacterialproduction of the protein. The following table shows a selection oftested plasmid/bacterial strain combinations for the expression of GDF-5related precursor proteins. Note that only constructs comprising a DNAcoding for an aminoterminal basic elongation of the precursor proteinled to the expression of precursor proteins.

Expression Bacterial +: good, Plasmid (SP = Signal Peptide) Strain +++:high pKOT (Tac-Promotor), +SP W3110 BP no pKOT (Tac-Promotor), +SP BL21(DE3) no pKOT (Tac-Promotor) , −SP W3110 BP no (37° C.) pKOT(Tac-Promotor), −SP W3110 BP no (22° C.) pKOT (Tac-Promotor), −SP BL21(DE3) Lys no pKOT (Tac-Promotor), −SP Origami (DE3) no pKOT(Tac-Promotor), −SP HMS174 (DE3) no pGEM (T7-Promotor), −SP BL21 (DE3)no pGEM (T7-Promotor), −SP JM109 No pET15b (T7-Promotor), Basic-tag BL21(DE3) Yes (+) pET15b (T7-Promotor), Basic-tag Rosetta Yes (+++)(Optimized Codon Usage) pKOT (Tac-Promotor), −SP Rosetta no pKOT(Tac-Promotor), +SP Rosetta No

The results disclosed herein demonstrate that said sequence extension isespecially beneficial if it comprises a continuous stretch of five ormore basic amino acids (arginine, histidine or lysine) which forces thebacterial cell to produce the protein.

In a preferred embodiment of this part of the invention, the N-terminusof such fusion protein comprises the sequence HHHHH (5× histidine).Especially preferred are precursor proteins which comprise a continuousstretch of 6 histidines. For reasons of precaution it is noted that saidbasic stretch is only needed for the protein production within thebacterial cell and is not required for already established industrialprotein purification techniques.

In other preferred embodiments, the N-terminus comprises the sequencesLLLLL, RRRRR, HLHLH or RHRHR.

If a fusion protein is generally unwanted, it is of course also possibleto modify the original protein sequence without addition of amino acids,i.e. by simply replacing a part of the original protein sequence by saidcontinuous stretch of five or more basic amino acids. However, it isrequired that such replacement should be done within the first 10 aminoacids of the original protein sequence.

Non-parenteral administration of a protein comprising an amino-terminalextension or a modification comprising a basic stretch of amino acidsinto mammals might create some immunogenic problems. To avoid anundesired immune response, it is helpful if the modified or added aminoacid sequence of the bacterially expressed GDF-5 related precursorprotein can be removed prior to the administration to mammalianpatients. Removal can be easily achieved by different techniques, e.g.if an adequate protease site (which is different from the protease siterequired for biological activation) is recombinantly introduced into theprotein sequence of the GDF-5 related precursor protein. In a preferredembodiment, said protease site is selected from the group consisting ofsites for thrombin, enterokinase, factor Xa or sumo protease.

As an alternative, the N-terminal extension of the GDF-5 precursorprotein might also be removed by an autocatalytic cleavage processinduced either by pH-shift or reducing agents such as DTT or betamercaptoethanol (see example 7). For example, the inducibleself-cleavage activity of protein splicing elements such as e.g. inteinscan be used to separate the GDF-5 precursor protein from the N-terminalaffinity tag.

For this purpose, the GDF-5 precursor protein can e.g. be integrated invectors such as the commercially available IMPACT-TWIN (Intein MediatedPurification with Affinity Chitin-binding Tag-Two Intein, New EnglandBiolabs) system. IMPACT-TWIN is able to isolate native recombinantproteins without the use of exogenous proteases. Intein1 is amini-intein derived from Synechocystis spdnaB gene and engineered toundergo pH and temperature dependent cleavage at its C-terminus (Mathyset al. (1999), Gene 231, 1-13). Intein2 is either a mini-intein from theMycobacterium xenopi gyrA gene (pTWIN1) or from the Methanobacteriumthermoautotrophicum rir1 gene (pTWIN2). These inteins have been modifiedto undergo thiol-induced cleavage at their N-terminus (Southworth etal., (1999) Biotechniques 27, 110-120). The use of thiol reagents suchas 2-mercaptoethanesulfonic acid (MESNA) releases a reactive thioesterat the C-terminus of the target protein.

It has additionally been found that the recombinant expression of saidGDF-5 related precursor protein in prokaryotes can be also enhanced byother genetic modifications. This enhancement requires that certain DNAtriplets encoding the amino acids isoleucine, arginine, leucine, prolineand glycine, which appear to be detrimental for the expression of therecombinant precursor proteins in bacteria, have to be removed from apart of the DNA coding for the GDF-5 related precursor protein.

More precisely, in another preferred embodiment of this part of theinvention, bacterial expression of a GDF-5 related precursor protein isclearly facilitated if triplets AUA (Ile), AGG, AGA, CGG, CGA (Arg), CUA(Leu), CCC (Pro) and GGA (Gly) have been replaced by alternativetriplets of the genetic code encoding identical amino acids. As shown inFIG. 5, such optimized codon usage has been implemented in a pET15b(T7-Promotor, Basic-tag) vector system. Expression of the precursorprotein in the E. coli strain Rosetta with optimized codon usageresulted in a high yield protein production. Use of a similar vectorsystem but without optimized codon usage in bacterial strain BL21(DE3)yielded lower amounts of the precursor protein.

In the most preferred embodiment of this part of the invention, acontinuous stretch of two or more of said detrimental triplets withinthe first 30 codons of said DNA molecule has to be avoided.

The yield and quality of the GDF-5 related precursor protein can befurther dramatically improved if the bacterial production methodcomprises optimized purification steps. It has been found out that bestresults are achieved if the purification process comprises a directrefolding step. Direct refolding means that the expressed proteins areused directly after inclusion body preparation in a refolding procedure(necessary for renaturation) without prior column purification step.Usually GDF-5 related proteins are column purified before initiation ofrefolding, see e.g. WO96/33215). Especially important for the discloseddirect refolding procedure is the use of an optimized buffer forsolubilization of inclusion bodies containing not more than 3 mM DTT. Ithas been found out that more DTT content interferes with the refoldingprocedure, which highly depends on a redox system which is sensitive toreducing agents like DTT. Due to the low amount of DTT, the refoldingstep can be performed in a 1:10 dilution instead of a commonly used1:100 dilution, thus having positive effects on the protein yield.Specific parameters of these preferred purification conditions aredisclosed in example 1.

GDF-5 related precursor proteins which are manufactured in bacteria bythe aforementioned methods have a variety of advantages besides theircost effective production. It is also essential that the recombinantprecursor proteins of the invention, although expressed in bacteria andtherefore lacking essential eukaryotic features such as e.g.glycosylation, can be proteolytically cleaved and activated in a mannercomparable to precursor proteins expressed in eukaryotes. Proteolyticcleavage of proteins belonging to the family of growth anddifferentiation factors often occurs at a characteristic RX(K/R)R sitethat divides the mature peptide from the amino-terminal prodomain. Forexample, the cleavage site of human GDF-5 contains the motif RRKR,whereas the corresponding sites of GDF-6 and GDF-7 contain the sequenceRRRR. These sites are known to be recognized by subtilisin likeproprotein convertases (SPCs), a family of seven structurally relatedserine endoproteases (designated SPC1 to SPC7). Although allsubtilisin-like proprotein convertases can be used for the cleavage ofthe precursor proteins of the invention, protease SPC1 (also designatedfurin) is especially useful. Also preferred are SPC4, SPC6 and SPC7because they are coexpressed with growth factor proteins at distinctsites (see Costam et al. 1996, J. Cell Biol. 134, 181-191). Especiallypreferred according to the invention is furthermore the addition ofsingle SPCs or combinations of different SPCs in pharmaceuticalcompositions comprising GDF-5 related precursor proteins. Suitablecombinations are e.g. SPC1 and SPC4, SPC1 and SPC6, and SPC1 and SPC7.Another preferred option is the cleavage of the precursor proteins withtrypsin (as shown in FIG. 7).

The extracellullar matrix is believed to serve as storage site forgrowth and differentiation factors. Thus, also suitable to releaseactive mature protein from the precursor proteins of the invention areother matrix proteases, e.g. matrix metalloproteases, preferably MMP3.

As a non-limiting example, FIG. 6 and example 3 show the cleavage of abacterially produced precursor protein of the invention (human GDF-5precursor) with proprotein convertase SPC1 (also designated furin). Inmammals, furin is predominantly localized within the trans-Golgi network(TGN)/endosomal system, but has also been detected on the cell surfaceand extracellularly (Molloy et al., 1999).

FIG. 7 and example 4 (ALP activity assay) demonstrate that the matureGDF-5 protein, which is released from the cleaved recombinant precursorby selected proteases furin, trypsin and MMP3, exhibits significantbiological activity, whereas the recombinant precursor protein itself isbiologically inactive.

The GDF-5-related precursor proteins as defined herein comprise a) aprotease site necessary for proteolytic activation and b) acysteine-knot-domain with an amino acid identity of at least 70% to the102 aa cysteine-knot domain of human GDF-5. Preferred are precursorproteins comprising a cysteine-knot domain with an amino acid identityof at least 75%, preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 95% to the 102 aacysteine-knot domain of human GDF-5. However, the limiting value of atleast 70% is well suitable to separate members of the GDF-5/-6/-7 groupof proteins as well as variants thereof from precursors of otherproteins such as other GDFs and other growth factors. A comparison ofthe 102 aa cysteine-knot-domains of human GDF-5, human GDF-6 and humanGDF-7 (FIG. 2) reveals the high grade of amino acid identity betweenthese proteins. Human GDF-6 shares 87 (85%) and human GDF-7 83 (81%)identical residues with the cysteine-knot-domain of human GDF-5. Incontrast, GDFs and BMPs not belonging to the GDF-5/-6/-7 subgroupdisplay much lower identity values below 60%.

The determination of corresponding amino acid positions in related aminoacid sequences as well as the calculation of percentages of identitybetween can be performed with the help of well known alignmentalgorithms and optionally computer programs using these algorithms. Theamino acid identities in this patent application have been calculated byaligning sequences with the freeware program ClustalX (Version 1.81)with default parameters and subsequent counting of identical residues byhand. Default settings for pairwise alignment (slow-accurate) are: gapopening parameter: 10.00; gap extension parameter 0.10; Protein weightmatrix: Gonnet 250. The ClustalX program is described in detail in:

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. andHiggins, D. G. (1997)

The ClustalX windows interface: flexible strategies for multiplesequence alignment aided by quality analysis tools.

Nucleic Acids Research 24:4876-4882.

ClustalX is a windows interface for the ClustalW multiple sequencealignment program and is i.e. available from various sources, i.e. byanonymous ftp from ftp-igbmc.u-strasbg.fr, ftp.embl-heidelberg.de,ftp.ebi.ac.uk or via download from the following webpage:http://www-igbmc.u-strasbg.fr/BioInfo/. The ClustalW program andalgorithm is also described in detail in:

Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994)

CLUSTALW: improving the sensitivity of progressive multiple sequencealignment through sequence weighting, positions-specific gap penaltiesand weight matrix choice. Nucleic Acids Research 22:4673-4680.

As already explained and shown in example 6 (FIG. 9, respectively), therecombinant precursor proteins of the invention are biologicallyinactive but can be activated in vivo/in situ by proteases. This isespecially advantageous in cases where precursor proteins orpharmaceutical compositions comprising said precursor proteins aretherapeutically administered to a patient. Since proteases like e.g.SPCs are detectable in the Golgi network but are also located in othercell compartments and the extracellular matrix in various amounts independency of the bodies need, they are able to convert administeredprotein precursors to active mature proteins by and by in a mannercontrolled by the mammalian metabolism. Thus, administration of proteinprecursors guarantees a sustained release of the active drug over alonger period and avoids dosage problems as known from theadministration of mature/active growth factors.

Although the administration of pure GDF-5 related precursor proteinsshould be sufficient for some therapeutic purposes, other indicationsmay require the administration in combination with protease formulationscomprising e.g. Trypsin, SPCs and matrix metalloproteases, for examplebecause some tissues are characterized by the lack of endogenousprotease production.

It should also be mentioned that SPCs and other proteases show distincttissue specific expression patterns. It is well known that some areubiquitously present whereas others are restricted to a certain tissue.Although the precursor proteins of the invention can be activated e.g.by SPC1/furin alone (see example 3), the tissue specific expressionshould be taken into account if pharmaceutical compositions comprisingprecursor proteins in combination with proteases are administered. Insome cases, precursor protein activation in some tissues may be enhancedif the pharmaceutical compositions comprise a protease with strongexpression pattern in said tissue. In other cases, two or more proteasesare beneficial for full activation. For example, it is known that SPC2,SPC3 and SPC4 expression is largely confined to neuronal tissues,whereas GDF-5 is activated in joints by a combination of two SPCs,namely SPC1 (Furin) and SPC6. SPC4 and SPC6 expression in developinglimbs overlaps with that of Growth and differentiation factors. SPC7seems to be ubiquitously expressed.

Additional expression patterns of SPCs are well known from variouspublications, see e.g. Seidah et al. 1990, DNA Cell Biol. 9, 415-424;Nakayama et al. 1992, J. Biol. Chem. 257, 5897-5900; Beaubien et al.1995, Cell Tiss. Res. 279, 539-549; Dong et al. 1995, J. Neurosci. 15,1778-1796). If desired, such expression patterns can also be easilydetermined by routine techniques such as in situ hybridization, antibodystaining, or RT-PCR.

In an especially preferred embodiment of this part of the invention, apharmaceutical composition comprising a precursor protein and a proteaseformulation is designed in a way which provides retarded release of saidproteases. This composition ensures that the precursor proteins of theinvention are activated at a time point when the administered solutionreaches the target site. There are countless methods for the sustainedrelease of proteins described in the art which can be used. For a reviewsee e.g. Handbook of Pharmaceutical Controlled Release Technology (Wise,D., ed.), 2000. For example, a slow-release formulation may compriseproteins bound to or incorporated into particulate preparations ofpolymeric compounds (such as polylactic acid, polyglycolic acid, etc.)or liposomes.

In another embodiment of this part of the invention, the proteaseformulation is administered separately a certain time period afteradministration of the precursor proteins of the invention.

A prerequisite for pharmaceutical compositions which are intended forparenteral administration is a nearly physiological pH of saidcompositions. Whereas mature GDF-like proteins such as GDF-5 showexceptional poor solubility under physiological conditions, thebacterially expressed precursor proteins of this invention demonstrate apH-dependant solubility profile which allows for the direct parenteraladministration to mammals. As exemplified in FIG. 8 and Example 5, theyexhibit excellent solubility between pH 6 and pH 8, and especiallyimportant at or around physiological pH 7.

Whereas mature GDF-5 related proteins are insoluble at physiological pHand the use of these proteins is therefore restricted to local delivery,e.g. in combination with solid matrix materials, the excellentsolubility predetermines the precursor proteins and pharmaceuticalcompositions of the invention for systemic delivery purposes. Thisallows for the efficient treatment of disorders and diseases withsystemic character. The most prominent examples for such a systemicdisease are osteoporosis, rheumatism and osteoarthritis. However, liquidpharmaceutical compositions such as those disclosed herein can also beefficiently used for local delivery, e.g. via injection. For example,the pharmaceutical compositions and precursor proteins of the inventionare useful for the treatment of neurodegenerative disorders such asParkinson's disease (e.g. via intracerebral infusion or intranasaldelivery), for the treatment of local osteoarthritis or arthrosis (e.g.via injection into the affected tissue, organ or joint), for theregeneration of meniscus and spinal disks (e.g. via injection), for thetreatment of hair loss and skin aging (e.g. via topical creams) etc. . ..

According to this novel characteristic, the precursor proteins andpharmaceutical compositions of the inventions can be administeredparenterally. Such parenterally administered therapeutic compositionsare typically in the form of pyrogen-free, parenterally acceptableaqueous solutions comprising the pharmaceutically effective component(s)in a pharmaceutically acceptable carrier and/or diluent. Said parenteraladministration might e.g. be dermal, ocular, pulmonary, topical orintranasal administration, injection or infusion.

Of course this invention also comprises pharmaceutical compositionscontaining further substances like e.g. pharmacologically acceptableauxiliary and carrier substances. The formulation may includeantioxidants, preservatives, colouring, flavouring and emulsifyingagents, suspending agents, solvents, fillers, bulking agents, bufferssuch as phosphate buffered saline (PBS) or HEPES, delivery vehicles,excipients and/or pharmaceutical adjuvants. For example, a suitablecarrier or vehicle may be water for injection, physiological salinesolution, or a saline solution mixed with a suitable carrier proteinsuch as serum albumin. A preferred antioxidant for the preparation ofthe composition of the present invention is ascorbic acid.

Cosmetic compositions known in the art, preferably hypoallergic and pHcontrolled are especially preferred, and include toilet waters, packs,lotions, skin milks or milky lotions. Said preparations contain, besidesthe active compound, components usually employed in such preparations.Examples of such components are oils, fats, waxes, surfactants,humectants, thickening agents, antioxidants, viscosity stabilizers,chelating agents, buffers, preservatives, perfumes, dyestuffs, loweralkanols, and the like. If desired, further ingredients may beincorporated in the compositions, e.g. antiinflammatory agents,antibacterials, antifungals, disinfectants, vitamins, sunscreens,antibiotics, or other anti-acne agents.

The solvent or diluent of the pharmaceutical composition may be eitheraqueous or non-aqueous and may contain other pharmaceutically acceptableexcipients which are capable of modifying and/or maintaining a pH,osmolarity, viscosity, clarity, scale, sterility, stability, rate ofdissolution or odour of the formulation. Similarily other components maybe included in the pharmaceutical composition according to the presentinvention in order to modify and/or maintain the rate of release of thepharmaceutically effective substance. Such modifying components aresubstances usually employed in the art in order to formulate dosages forparenteral administration in either unit or multi-dose form. The finallyformulated pharmaceutical and/or diagnostic composition preparedaccording to the present invention may be stored in sterile vials inform of a solution, suspension, gel, emulsion, solid or dehydrated orlyophilized powder. These formulations may be stored either in aready-to-use form or in a form, e.g. in case of a lyophilized powder,which requires reconstitution prior to administration. The above andfurther suitable pharmaceutical formulations are known in the art andare described in, for example, Gus Remington's Pharmaceutical Sciences(18th Ed., Mack Publishing Co., Eastern, Pa., 1990, 1435-1712). Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the pharmaceuticallyeffective component.

The pharmaceutical composition may comprise a matrix material, i.e. incases where regeneration of bone or cartilage is intended. It isadvantageous to the precursor proteins when they are applied in and/oron a biocompatible matrix material. Matrix material as used herein meansa carrier or matrix acting as a scaffold for cell recruitment,attachment, proliferation and differentiation and/or as a potentialdelivery and storage device for precursor proteins. In contrast to thesolid matrices, carriers consist of amorphous materials having nodefined surfaces and lacking a specific shape, i.e. alkylcelluloses,pluronics, gelatins, polyethylene glycols, dextrins, vegetable oils,sugars and other liquid and viscous substances.

Uses of growth factors in combination with matrix materials areextensively published and described, such as for example in WO98/21972.These matrix materials are equally suitable for precursor proteins ofgrowth factors according to the present invention. The matrix materialcan be transplanted into the patient, e.g. surgically, wherein theprotein or the DNA encoding the protein can be slowly released from thematrix material and then be effective over a long period of time. Alltypes of matrix materials are useful in accordance with the presentinvention, as long as they are biocompatible and selected for theintended area or indication of use. The matrix material can be a naturalmaterial, a modified natural material as well as a synthetic material.All already known matrices for morphogenetic proteins are encompassed.Examples of natural materials are e.g. autologous, heterologous orxenologous bone materials, collagen, e.g. collagen type I and III, ormetals like titanium. Also other components of the extracellular matrixcan be used. The extracellular matrix comprises for example the variouscollagens, as for example types I, II, V, IX, X, XI and XIII, furtherproteoglycanes and glycosaminoglycanes, as for examplechondroitinsulfate, biglycane, decorine and/or hyaluronic acid, ornoncollagenous proteins as for example osteopontin, laminin,fibronectin, vitronectin, thrombospondin, cartilage matrix protein anddentin phosphoprotein. All mentioned natural materials may also be usedin artificially modified forms. Examples of modified natural materialsare demineralized bone, thermoashed bone mineral, sintered bone orchemically crosslinked hyaluronic acid (hydrogel), or metal alloys.Examples of synthetic materials are polymers like polyglycolic acid,polylactide and polylactide derivatives such as e.g. polylactic acid,poly(lactide-co-glycolide), polylactid acid-polyethylene glycol orglycolide L-lactide copolymers, further polyphosphates, polyethyleneglycol, polyoxyethylene polyoxypropylene copolymers or materialscontaining calcium phosphates such as beta-tricalcium phosphate(Ca₃(PO₄)2), alpha-tricalcium phosphate and hydroxyl apatite. Furtherexamples of other useful matrix materials belonging to one of the abovementioned groups are Ca(OH)2, coral, natural bone mineral, chitin,non-demineralized bone particles, ceramic bone particles, ceramicdentin, irradiated cancellous bone chips, plaster of Paris, bioactiveglass, apatite-wollastonite-containing glass ceramic. Also a combinationof the above mentioned carriers and/or matrices can form the matrixmaterial as for example the combination of hydroxy apatite and collagen(e.g. Healos, previously available from Orquest, Inc., CA, USA, [nowDePuy Acromed, MA, USA]), a combination of polyglycolic acid andpolylactic acid or polylactid derivatives, or coral-collagen composites.For a non limiting list of useful carriers and matrices see further i.e.Kirker-Head 2000, Advanced Drug Delivery 43, 65-92.

In general, the precursor proteins or pharmaceutical compositionsthereof can be applied wherever mature recombinant and wild-type GDF-5forms have been successfully used. For example, mature GDF-5 isconsidered to be a very effective promoter of bone and cartilageformation as well as connective tissue formation (see for example WO95/04819, Hötten et al. 1996, Growth Factors 13, 65-74; Storm et al.1994, Nature 368, 639-643; Chang et al. 1994, J. Biol. Chem. 269,28227-28234) and formation of connective tissue attachment (EP 0 831884. In this context, GDF-5 is useful for applications concerning thejoints between skeletal elements (see for example Storm & Kingsley 1996,Development 122, 3969-3979). One example for connective tissue is tendonand ligament (Wolfman et al. 1997, J. Clin. Invest. 100, 321-330;Aspenberg & Forslund 1999, Acta Orthop Scand 70, 51-54; WO 95/16035).The protein is helpful for meniscus and spinal/intervertebral diskrepair (Walsh et al. 2004, Spine 29, 156-63) and spinal fusionapplications (Spiro et al. 2000, Biochem Soc Trans. 28, 362-368). GDF-5can be beneficially applied in tooth (dental and periodontal)applications (see for example WO 95/04819; WO 93/16099; Morotome et al.1998, Biochem Biophys Res Comm 244, 85-90) such as the regeneration ofdentin or periodontal ligament.

Mature GDF-5 is also useful in wound repair of any kind. It is alsobeneficial for promoting tissue growth in the neuronal system andsurvival of e.g. dopaminergic neurons. In this context, GDF-5 can beused for treating neurodegenerative disorders like e.g. Parkinson'sdisease and possibly also Alzheimer's disease or Huntington choreatissues (see for example WO 97/03188; Krieglstein et al., (1995) J.Neurosci Res. 42, 724-732; Sullivan et al., (1997) Neurosci Lett 233,73-76; Sullivan et al. (1998), Eur. J. Neurosci 10, 3681-3688). GDF-5allows to maintain nervous function or to retain nervous function inalready damaged tissues. GDF-5 is therefore considered to be a generallyapplicable neurotrophic factor. If e. g. nerve guide carriers are coatedwith GDF-5 or precursor proteins thereof, significant healing of nervedamages of the peripheral nervous systems can be anticipated.

The precursor proteins and pharmaceutical compositions of the inventioncan also be used for prevention or therapy of damage of periodontal ordental tissue including dental implants, neural tissue including CNStissue and neuropathological situations, tissue of the sensory system,liver, pancreas, cardiac, blood vessel, renal, uterine and thyroidtissue, skin, mucous membranes, endothelium, epithelium, for promotionor induction of nerve growth, tissue regeneration, angiogenesis, woundhealing including ulcers, burns, injuries or skin grafts, induction ofproliferation of progenitor cells or bone marrow cells, for maintenanceof a state of proliferation or differentiation for treatment orpreservation of tissue or cells for organ or tissue transplantation; forintegrity of gastrointestinal lining, for treatment of disturbances infertility, contraception or pregnancy.

It is also useful for diseases of the eye, in particular retina, corneaand optic nerve (see for example WO 97/03188; You et al. (1999), InvestOpthalmol Vis Sci 40, 296-311), for hair growth and the treatment anddiagnosis of skin related disorders (WO 02/076494; Battaglia et al.2002, Trans. Orthop. Res. Soc. 27, 584), and for induction ofangiogenesis (Yamashita et al. 1997, Exp. Cell Res. 235, 218-26).

Diseases concerning sensory organs like the eye are also to be includedin the preferred indication of the pharmaceutical composition accordingto the invention. As preferred neuronal diseases again Parkinson's andAlzheimer's diseases can be mentioned as examples.

The following non-limiting examples together with the figures andsequence protocols are intended to further illustrate the invention.

SEQ ID NO: 1 shows the protein sequence of the human GDF-5 precursor.

SEQ ID NOs: 2 to 4 show the amino acid sequences of the cysteine domainsof GDF-6, GDF-7 and GDF-5, respectively.

SEQ ID NO: 5 shows the amino acid sequence of the recombinant humanGDF-5 precursor shown in FIG. 4.

FIG. 1 shows additional features of the human GDF-5 precursor proteinaccording to SEQ ID NO: 1:

aa 001-381 pre-prodomain (bold letters) aa 001-027 signal peptide (boldand underlined) aa 382-501 mature protein part aa 400-501cysteine-knot-domain (underlined)

FIG. 2 shows a comparison of the 102 aa cysteine-knot-domains of humanGDF-5 (SEQ ID NO: 4), human GDF-6 (SEQ ID NO: 2; sequence 2 from U.S.Pat. No. 5,658,882) and human GDF-7 (SEQ ID NO: 3; sequence 26 from U.S.Pat. No. 5,658,882). Amino acid residues which are identical in allthree molecules are highlighted in black.

FIG. 3 shows a table with the sequence identities ofcysteine-knot-domains of known BMPs and GDFs to the cysteine-knot-domainof human GDF-5.

FIG. 4 shows the amino acid sequence (SEQ ID NO: 5) of a rhGDF-5precursor protein construct (proGDF-5) without signal peptide, butcomprising an aminoterminal extension including basic stretch andthrombin cleavage site according to example 1. His-tag: bold underlined.Thrombin cleavage recognition site: underlined. rhGDF-5 pro-domainwithout signal peptide: boxed. Mature rhGDF-5: bold. Furin recognitionsite: shaded.

FIG. 5 shows a Western blot of GDF-5 precursor protein (proGDF-5, seeFIG. 4) expression (see example 2) in E. coli. Lane 1 and 2: rhGDF-5precursor protein expression in E. coli strain BL21(DE3). Lane 3 and 4:rhGDF-5 precursor protein expression in E. coli strain Rosetta. C:mature rhGDF-5 positive control.

FIG. 6 shows SDS PAGE analysis of rhGDF-5 precursor protein after furindigestion by SDS PAGE.

Lane 1: rhGDF-5 precursor protein after furin digestion, release ofmature GDF-5 (arrow with double line). Lane 2: rhGDF-5 precursor protein(bold arrow). Lane 3: mature rhGDF-5 positive control.

FIG. 7 shows ALP results (average values of 3 independent experiments)which are confirming proteolytical activation of rhGDF-5 precursorprotein (proGDF-5) after digestion with furin, trypsin or MMP3 (control:undigested precursor protein) according to example 4. Six differentprotein concentrations (14.6 ng/mL, 44.5 ng/mL, 133.2 ng/mL, 400 ng/mLan 1200 ng/mL) have been used in this assay.

FIG. 8 shows solubility data of rhGDF-5 precursor protein according toexample 5 at pH 2, pH 7 and pH8.

FIG. 9 shows the activation of GDF-5 precursor protein in chickenmicromass cultures as described in example 6. The induction of cartilageproduction is indicated by the increase in Alcian blue staining. Bothmicromass cells incubated with mature rhGDF5 as well as with rhGDF5precursor protein showed a massive increase of cartilage, indicating thein vivo cleavage/activation of the recombinant precursor protein outsideof the trans-golgi network.

EXAMPLE 1 Creation, Expression and Purification of rhGDF-5 PrecursorProteins

rhGDF-5 (recombinant human GDF-5) precursor protein without signalpeptide was integrated into the protein expression vector pET15b(Novagen) using the restriction sites NdeI and BamHI. The vector codesfor an N-terminal histidine tag and a thrombin cleavage site. Theexpression of a rhGDF-5 precursor construct (proGDF-5, see FIG. 4) wasperformed in the E. coli strains BL21(DE3) and Rosetta (Novagen).Rosetta is a BL21 derivative to enhance the expression of eukaryoticproteins that contain codons rarely used in E. coli the strain supplytRNAs for the codons AUA, AGG, AGA, CUA, CCC, CGG and GGA on achloramphenicol-resistant plasmid. The protein expression was inducedwith IPTG, the proteins were expressed in inclusion bodies. Theseinclusion bodies were isolated using a homogenization buffer (25 mM TrisHCl, 10 mM EDTA, pH 7.3) and wash buffer (20 mM Tris HCl, 5 mM EDTA, pH8.3) according to standard procedures. The inclusion bodies weresolubilized in solubilization buffer (4 M GuHCl, 3 mM DTT, 0.1 M TrisHCl, pH 8.5) and the direct refolding was performed in a 1:10 dilutionwith refolding buffer (1 M Ariginine-HCl, 5 mM oxidized glutathione(GSSG), 1 mM reduced glutathione (GSH), 0.1 M Tris HCl, 5 mM EDTA, pH8.0) at room temperature for 5 days. Ultrafiltration was conducted toadjust the buffer terms and to reduce the volume to an optimum load forthe SEC-column. Buffer exchange was performed from 100 mM Tris HCl, 50mM EDTA, pH 8.0 to 2 M Urea, 100 mM Tris, 150 mM NaCl, 50 mM EDTA, pH8.0. Equipment and conditions (Ultrafiltration stiring cell; AmiconModel 8050 (50 mL), Membrane; Pall Filtron 30 kD, OM030076,Gas/Pressure; N₂/max. 4 bar). Further purification was carried out on asize exclusion column (GE Healthcare Amersham Biosciences, hiLoad 26/60;column material: Superdex 200 prepgrade, column volume 319 mL) flow rate2.5 mL/min. 6 mL protein from the ultrafiltration procedure was loadedon the column. Protein was eluted in 2 M urea, 100 mM Tris HCl, 5 mMEDTA, pH 8.0. An additional purification step was carried out on reversephase HPLC (GE Healthcare Amersham Biosciences, column HR16/10; columnmaterial Source 15RPC volume 20 mL), flow rate 3 mL/min, System: ÄktaEplorer 100. Gardient was started with 35% of Eluent B (0.1% TFA, 90%CH3N, HPLC H₂O) then gradient from 35% to 60%, slope 0.38%/min, thengradient 60% to 90%, slope 1.5%/min, then 90% two column volumes andfinally 35%, two column volumes. The fractions containing the dimerizedprotein were pooled, lyophilized and stored at −80° C.

EXAMPLE 2 Analysis of rhGDF-5 Full Length Protein Expression by WesternBlotting

E. coli strains BL21 (DE3) and Rosetta were transformed with the plasmidpET15b-rhGDF-5 full length. Protein expression was induced with IPTG.Bacterial pellets were dissolved in SDS sample buffer and separatedunder reducing conditions on a 16% acrylamid SDS gel. The proteins wereblotted onto PVDF membrane and detected with a chemiluminescencedetection kit (Applied Biosystems), using the polyclonal antibody AntirhGDF-5 (Chicken B Pool).

Protein expression of rhGDF-5 precursor protein (GDF-5pro) could only bedetected after IPTG induction. In comparison to the BL21(DE3) anincreased protein expression could be achieved in the Rosetta strain.The improved protein expression in Rosetta might be due to the optimizedcodon usage. Protein expression was optimized with different expressionvector system and different E. coli strains. Surprisingly, precursorprotein expression was only possible when rhGDF-5 precursor had anadditional N-terminal protein tag e.g. histidine tag. Protein expressionof rhGDF-5 full length without N-terminal modification was not possible.

EXAMPLE 3 Cleavage of GDF-5 Precursor Protein by Furin

The in vitro digestion of rhGDF-5 precursor protein was performed withthe specific proprotein convertase furin. Furin cleaves the amino acidrecognition sequence R-K-R-R within the rhGDF-5 full length. A typicalcleavage experiment was carried out with 3 μg rhGDF-5 full lengthdissolved in 1× PBS supplemented with 1 mM CaCl₂ and incubated with 3 UFurin (New England Biolabs) at 30° C. over night. The digestion wascontrolled by Coomassie stained SDS gels and Western blot analysis withantibodies directed against the mature rhGDF-5. RhGDF-5 full length wasdigested with furin and separated under non-reducing conditions on a 10%acrylamid SDS gel (see FIG. 6). The proteins were further blotted ontoPVDF membrane and detected via western blotting with a chemiluminescencedetection kit (Applied Biosystems), using the mouse monoclonal antibodyaMP-5.

The rhGDF-5 full length has a molecular weight of ca. 100 kDa. Afterdigestion with furin the mature GDF-5 was released. In the western blot,aMP-5 antibody only detects correctly folded GDF-5, therefore it isdemonstrated that furin generates mature rhGDF-5 from its naturalprecursor protein rhGDF-5 full length.

EXAMPLE 4 Measurement of the Biological Activity of rhGDF-5 PrecursorProtein In Vitro by ALP Assay

5×10⁵ cells of mouse stromal MCHT-1/26 cells were incubated for 3-4 daysin 20 ml cell culture medium (alpha-MEM, Penicilline/Streptomycine, 2 mML-glutamine, 10% FCS) at 37° C., 5% CO₂, H₂O-saturated. The cells weresubsequently washed with PBS (phosphate buffered saline), trypsinatedand resuspended in culture medium to a density of 3×10⁴ cells/ml. 150 μlwere transferred to each well of a 96 well culture plate and incubatedfor 24 h at 37° C., 5% CO₂, H₂O-saturated. After washing with medium thewells were filled with 120 μl of new culture medium. 40 μl of differentdilutions of rhGDF-5 full length and rhGDF-5 for standard curve(dissolved in 10 mM HCl and diluted at least 250 fold in medium) wereadded, followed by another incubation step for 72 h at 37° C., 5% CO₂,H₂O-saturated. After washing with PBS, 150 μl of lysis solution (0.2%Nonidet P40, 0.2 g MgCl₂×6H₂O, adjusted to 1000 ml with water) wasadded, followed by 15-18 h incubation at 37° C., 5% CO₂, H₂O-saturated.50 μl of each well were subsequently transferred to a new 96 well plate.50 μl of substrate solution (2.5× concentrated diethanolamine substratebuffer+148 g/I PNPP (sodium p-nitrophenyl-phosphate) was then added toeach well and the plates were incubated for another 60 min at 37° C., 5%CO₂, H₂O-saturated. The ALP-reaction was stopped afterwards with 100 μlof 30 g/I NaOH and finally the optical density was measured with anautomatic microplate reader at 405 nm under consideration of blank valuesubtraction. As an example, results (average values of 3 independentexperiments) regarding rhGDF-5 precursor protein either digested withfurin or undigested are shown in FIG. 7. Six different proteinconcentrations (14.6 ng/mL, 44.5 ng/mL, 133.2 ng/mL, 400 ng/mL an 1200ng/mL) have been used in this assay. The undigested rhGDF-5 precursorprotein protein exhibits nearly no biological activity. In contrastrhGDF-5 full length digested with furin, trypsin or MMP-3 exhibitbiological activity in a dose dependent manner. The protease furin alone(as a control) has no negative influence on the ALP assay. Therefore wecan conclude that rhGDF-5 precursor protein is a pro-form exhibiting nodetectable biological activity in the alkaline Phosphatase assay. ALPinduction of rhGDF.5 precursor protein depends on a proteolyticalactivation.

EXAMPLE 5 Solubility of rhGDF-5 Precursor Protein

Chromatography purified rhGDF-5 full length was eluted from a sizeexclusion column in 100 mM Tris HCl, 5 mM EDTA, pH 8.0 buffer. Todetermine the solubility protein solution (1.2 mg/mL) was adjusted withHCL to pH 2.0 and pH 7.0. Subsequently the protein solutions werecentrifuged 13.000 g for 10 minutes. The supernatant was carefullyremoved and the pellet was solved in 10 μl SDS sample buffer. Thesupernatant and the pellet were separated on a 10% acrylamid SDS gel andanalyzed by gel densitometry (Aida, Version 3.51)

The rhGDF-5 full length showed a solubility of 99% in a buffercomprising of 100 mM Tris HCl, 5 mM EDTA, for the pH values 2, 7, and 8.As an example for buffer with pH 7.0, 1.03 μg/100 μl protein was foundin the pellet and 98 μg/100 μL was found in the supernatant. Thereforeit can be concluded that rhGDF-5 full length is soluble at aphysiological pH.

EXAMPLE 6 Mimicked In Vivo Activation of Protein Precursors

The micromass system is intended to mimic the initial conditions thatlead to cartilage deposition in vivo. Primary cultures ofundifferentiated mesenchyme cells of limb buds reproduce cartilagehistogenesis, a fundamental step in the morphogenesis of the skeleton.Thus, in the micromass system, limb bud cells will form foci ofdifferentiating chondrocytes. In order to determine a potential cleavageof the rhGDF-5 precursor protein and formation of biologically activemature protein outside the trans-Golgi network, we used chickenmicromass cultures and measured cell differentiation and cartilaginousmatrix production.

Micromass cultures were prepared as described previously (Lehmann etal., Proc. Natl. Acad. Sci. U.S.A. 100 (2003), 2277-12282) with minormodifications. Briefly, fertilized chicken eggs were obtained fromTierzucht Lohmann and incubated at 37.5° C. in a humidified eggincubator for about 4.5 days. Ectoderm was removed, and cells wereisolated from the limb buds at stage HH23-24 by digestion with 0.1%collagenase type Ia and 0.1% trypsine. Micromass cultures were plated ata density of 2×10⁵ cells/10-μl drop. Cells were stimulated with theproteins rhGDF-5 and rhGDF-5 precursor protein (proGDF5) respectively;increasing protein concentrations ranging from 0 to 18 nM were applied.Culture medium (DMEM-F12, 2% chicken serum, 4 mM I-glutamine, 1000 U/mlpenicillin, and 100 μg/ml streptomycin) was replaced every 2 days.Alcian blue incorporation into the extracellular matrix of micromasscultures reflecting the production of proteoglycan-rich cartilaginousmatrix measured at day 4 was quantified after extraction. Alcian bluestaining was performed by fixing micromass cultures at day 4, thenincubating with 0.1% Alcian blue, pH 1, overnight. Quantification of thestaining was achieved after extensive washings with water by extractionwith 6 M guanidine-HCl for 8 hours at room temperature. Dyeconcentration was determined spectrophotometrically at A595.

As a result, both micromass cells incubated with mature rhGDF5 as wellas with rhGDF5 precursor protein showed a massive induction of cartilageproduction as indicated by the increase in Alcian blue staining (FIG.9), indicating the in vivo cleavage/activation of the recombinantprecursor protein outside of the trans-golgi network.

EXAMPLE 7 Removal of Aminoterminal Protein Extensions

The N-terminal extension of the GDF-5 precursor protein are removable byproteolytic processing with proteases such as thrombin, Factor Xa,enterokinase etc. Alternatively the N-terminal extension can beeliminated by autocatalytic cleavage processes induced either bypH-shift or reducing agents such as DTT or beta mercaptoethanol. Forthis purpose the GDF-5 precursor protein could be integrated in to theIMPACT-TWIN (Intein Mediated Purification with Affinity Chitin-bindingTag-Two Intein) system (New England Biolabs). This system utilizes theinducible self-cleavage activity of protein splicing elements termedinteins to separate the GDF-5 precursor protein from the N-terminalaffinity tag. These inteins have been modified to undergo thiol-inducedcleavage at their N-terminus. The use of thiol reagents such as2-mercaptoethanesulfonic acid (MESNA) releases a reactive thioester atthe C-terminus of the target protein.

7a) pH Induced Cleavage:

The rhGDF-5 precursor protein was cloned into a appropriate vector (e.g.pTWIN, containing the Ssp DnaB self-cleavable intein-tag). The resultingplasmid was transformed into an applicable E. coli host strain (i.e.ER2566, BL21). The cells were grown at 37° C. until an OD600 of 0.5-0.7was reached, protein induction was induced with IPTG. For column proteinpurification a chitin column was equilibrated with Buffer B1 (20 mMTris-HCl, pH 8.5, 100 mM NaCl, 1 mM EDTA). The cells were lysed inBuffer B1 and the clarified cell extract was slowly applied to thechitin column. The column was washed with Buffer B1 to remove theunbound proteins. The on-column cleavage of the intein-tag was inducedby equilibrating the chitin resin in Buffer B2 (20 mM Tris-HCl, pH 7.0,100 mM NaCl, 1 mM EDTA). To allow cleavage, the reaction was carried outovernight at room temperature. Finally the protein was eluted from thecolumn.

7b) Thiol-Induced Cleavage:

The rhGDF-5 precursor protein was cloned into a appropriate vector (e.g.pTWIN, containing either the Mxe GyrA or Mth RIR1 intein self-cleavableintein-tag). The resulting plasmid was transformed into an applicable E.coli host strain (i.e. ER2566, BL21). The cells were grown at 37° C.until an OD600 of 0.5-0.7 was reached, protein induction was inducedwith IPTG. For column protein purification a chitin column wasequilibrated with Buffer B2 (20 mM Tris-HCl, pH 7.0, 100 mM NaCl, 1 mMEDTA). The cells were lysed in Buffer B2 and the clarified cell extractwas slowly applied to the chitin column. The column was washed withBuffer B2 to remove the unbound proteins. The on-column cleavage of theintein-tag was induced by equilibrating the chitin resin in Buffer B3(20 mM Tris-HCl, pH 8.5, 100 mM NaCl, 40 mM DTT, 1 mM EDTA). To allowcleavage, the reaction was carried out overnight at room temperature.Finally the protein was eluted from the column with Buffer B3.

1-32. (canceled)
 33. Recombinant mammalian precursor protein or variantthereof comprising a) a protease site necessary for proteolytic cleavageand liberation of a biologically active mature GDF-5 related protein,and b) a cystine-knot domain with an amino acid identity of at least 70%to the 102 aa cystine-knot domain of human GDF-5 (amino acids 400-501 ofFIG. 1/SEQ ID NO 1); characterized in that said precursor protein isnon-glycosylated and produced in prokaryotes.
 34. Precursor proteinaccording to claim 33, said protein comprising the amino acids 28-501 ofthe sequence shown in SEQ ID NO: 1 GDF-5 precursor without signalpeptide).
 35. Precursor protein according to claim 33, said proteinhaving an alanine residue at position 4435 instead of a cysteine.(Monomeric GDF-5 precursor without signal peptide)
 36. Precursor proteinaccording to claim 33, said protein further having an aminoterminalextension of at least seven amino acids.
 37. Precursor protein accordingto claim 33, said protein comprising a recombinantly introducedcontinuous stretch of five or more basic amino acids (arginine,histidine or lysine).
 38. Precursor protein according to claim 36, saidprotein further comprising a) a recombinantly introduced protease siteintended for removal of said aminoterminal extension; or b) arecombinantly introduced site intended for pH-induced or reducingagent-induced removal of said aminoterminal extension.
 39. Precursorprotein according to claim 38 a), said protease site selected from thegroup consisting of sites for thrombin, enterokinase, factor xa or sumoprotease.
 40. A DNA molecule encoding a precursor protein according toclaim 33, characterized in that the triplets AUA (Ile), AGG, AGA, CGG,CGA (Arg), CUA (Leu), CCC (Pro) and GGA (Gly) within the first 30 codonshave been replaced by alternative triplets of the genetic code encodingidentical amino acids.
 41. A DNA molecule encoding a precursor proteinof claim 33, characterized in that stretches of at least two tripletsselected from the group consisting of AUA (He), AGG, AGA, CGG, CGA(Arg), CUA (Leu), CCC (Pro) and GGA (Gly) within the first 30 codonshave been replaced by alternative triplets of the genetic code encodingidentical amino acids.
 42. A process of manufacturing the recombinanteukaryotic precursor protein of claim 33 in prokaryotic host cells. 43.The process according to claim 42, characterized in that said processcomprises a step of transforming a prokaryotic host cell with a plasmidcontaining a DNA molecule encoding a precursor protein, characterized inthat the triplets AUA (Ile), AGG, AGA, CGG, CGA (Arg), CUA (Leu), CCC(Pro) and GGA (Gly) within the first 30 codons have been replaced byalternative triplets of the genetic code encoding identical amino acids.44. The process according to claim 42, further comprising solubilizationof inclusion bodies in a solution comprising 3 mM DTT or less.
 45. Theprocess according to claim 44, further comprising a direct refoldingprocedure which comprises a step of diluting the inclusion body solution10 fold or less.
 46. Pharmaceutical composition comprising one or moreproteins according to claim
 33. 47. Pharmaceutical composition accordingto claim 46, characterized in that said composition is soluble at a pHbetween 6 and
 8. 48. Pharmaceutical composition according to claim 47,characterized in that said composition is soluble at pH
 7. 49.Pharmaceutical composition according to claim 46, said compositionfurther comprising a protease formulation necessary for proteolyticrelease of the mature protein.
 50. Pharmaceutical composition accordingto claim 33, said protease formulation comprising one or more proteasesselected from the group consisting of matrix proteases and subtilisinlike proprotein convertases.
 51. Pharmaceutical composition according toclaim 49, said protease formulation providing retarded release of saidproteases.
 52. Pharmaceutical composition according to claim 49,characterized in that said are selected from the group consisting offurin, SPC-4 and SPC-6.
 53. Pharmaceutical composition according toclaim 49, characterized in that said protease formulation comprises acombination of either SPC-1 (furin) and SPC-6, SPC-1 (furin) and SPC-4,or SPC-1 (furin) and SPC-7.
 54. Protein according to claim 33 in theform of a depot formulation which is fully activated inside a mammalianbody at the target site by endogenous or co-administered proteases. 55.Pharmaceutical composition according to claim 46 for parenteraladministration.
 56. Pharmaceutical composition according to claim 55,wherein said parenteral administration is an injection or intracerebralinfusion.
 57. Pharmaceutical composition according to claim 55, whereinsaid parenteral administration is dermal, ocular, pulmonary, topical orintranasai administration.
 58. A method for the diagnosis, preventionand/or therapy of diseases associated with bone and/or cartilage damage,for promoting cartilage and/or bone formation and/or spinal fusion, forthe diagnosis, prevention and/or therapy of damaged or diseased tissueassociated with connective tissue including tendon and/or ligament,periodontal or dental tissue including dental implants, neural tissueincluding CNS tissue and neuropathological situations, tissue of thesensory system, liver, pancreas, cardiac, blood vessel, renal, uterineand thyroid tissue, skin, mucous membranes, endothelium, epithelium, forthe induction of nerve growth, tissue regeneration, angiogenesis, woundhealing including ulcers, burns, injuries and/or skin grafts, for theinduction of proliferation of progenitor cells and/or bone marrow cells,for maintenance of a state of proliferation or differentiation fortreatment or preservation of tissue or cells for organ or tissuetransplantation, for the treatment of degenerative disorders concerningthe joints to skeletal elements and/or for meniscus and/orspinal/intervertebral disk repair, for the manufacture of a therapeuticand/or diagnostic composition for the prevention and/or treatment ofneurodegenerative disorders in a patient in need of such method,comprising administering to said patient an effective amount of aprotein of claim
 33. 59. A method according to claim 58, wherein saidneurodegenerative disorder is selected from the group consisting ofParkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis(ALS), Multiple sclerosis and Huntington's disease.
 60. A methodaccording to claim 58, wherein said disease associated with bone andcartilage damage is osteoporosis.
 61. A method according to claim 58,wherein the method is for promoting hair growth.
 62. A method fordelivering a GDF-5 related precursor protein to a target site inside ofa mammal, comprising a) a first step of administration of a proteinaccording to claim 33, and b) a second independent step ofadministration of one or more proteases selected from the groupconsisting of Trypsin, matrix proteases and subtilisin like proproteinconvertases to the same target site.
 63. A method for delivering a GDF-5related precursor protein to the central and/or peripheral nervoussystem of a mammal, comprising administering to the mammal a proteinaccording to claim
 33. 64. A method for systemic delivery of GDF-5related precursor protein to a mammalian body, comprising administrationto the mammalian body of a protein according to claim
 33. 65. Theprocess according to claim 42, characterized in that said processcomprises a step of transforming a prokaryotic host cell with a plasmidcontaining a DNA molecule encoding said precursor protein, characterizedin that stretches of at least two triplets selected from the groupconsisting of AUA (He), AGG, AGA, CGG, CGA (Arg), CUA (Leu), CCC (Pro)and GGA (Gly) within the first 30 codons have been replaced byalternative triplets of the genetic code encoding identical amino acids.